Identification of kynurenine and quinolinic acid as promising serum biomarkers for drug-induced interstitial lung diseases

Background Drug-induced interstitial lung disease (DILD) is a lung injury caused by various types of drugs and is a serious problem in both clinical practice and drug development. Clinical management of the condition would be improved if there were DILD-specific biomarkers available; this study aimed to meet that need. Methods Biomarker candidates were identified by non-targeted metabolomics focusing on hydrophilic molecules, and further validated by targeted approaches using the serum of acute DILD patients, DILD recovery patients, DILD-tolerant patients, patients with other related lung diseases, and healthy controls. Results Serum levels of kynurenine and quinolinic acid (and kynurenine/tryptophan ratio) were elevated significantly and specifically in acute DILD patients. The diagnostic potentials of these biomarkers were superior to those of conventional lung injury biomarkers, Krebs von den Lungen-6 and surfactant protein-D, in discriminating between acute DILD patients and patients with other lung diseases, including idiopathic interstitial pneumonia and lung diseases associated with connective tissue diseases. In addition to identifying and evaluating the biomarkers, our data showed that kynurenine/tryptophan ratios (an indicator of kynurenine pathway activation) were positively correlated with serum C-reactive protein concentrations in patients with DILD, suggesting the potential association between the generation of these biomarkers and inflammation. Our in vitro experiments demonstrated that macrophage differentiation and inflammatory stimulations typified by interferon gamma could activate the kynurenine pathway, resulting in enhanced kynurenine levels in the extracellular space in macrophage-like cell lines or lung endothelial cells. Extracellular quinolinic acid levels were elevated only in macrophage-like cells but not endothelial cells owing to the lower expression levels of metabolic enzymes converting kynurenine to quinolinic acid. These findings provide clues about the molecular mechanisms behind their specific elevation in the serum of acute DILD patients. Conclusions The serum concentrations of kynurenine and quinolinic acid as well as kynurenine/tryptophan ratios are promising and specific biomarkers for detecting and monitoring DILD and its recovery, which could facilitate accurate decisions for appropriate clinical management of patients with DILD. Supplementary Information The online version contains supplementary material available at 10.1186/s12931-023-02653-6.

Serum TRP concentrations among the sample groups are shown in the box-and-whisker plot.The results of the statistical comparisons among the groups are summarized in Table 2. DAD/DAD-mixed: DILD patients in acute phase with CT pattern of diffuse alveolar damage, OP: DILD patients in acute phase with CT pattern of organizing pneumonia, NISP: DILD patients in acute phase with CT pattern of nonspecific interstitial pneumonia: Other: DILD patients in acute phase with CT pattern other than DAD, OP and NSIP, DILD recovery: patients recovered from DILD, DILD-tolerant: the patient group taking similar medications to the DILD group but without DILD onset, BP: bacterial pneumonia, NTM: nontuberculous mycobacteriosis, IIPs: idiopathic interstitial pneumonias, CTD: lung disease associated with connective tissue disease, COPD: chronic obstructive pulmonary disease, BA: bronchial asthma, HC: healthy control.S1 and S2, and the results of the statistical comparisons among the groups are summarized in Table S5.DAD/DAD-mixed: DILD patients in acute phase with CT pattern of diffuse alveolar damage, OP: DILD patients in acute phase with CT pattern of organizing pneumonia, NISP: DILD patients in acute phase with CT pattern of nonspecific interstitial pneumonia: Other: DILD patients in acute phase with CT pattern other than DAD, OP and NSIP, DILD recovery: patients recovered from DILD, DILD-tolerant: the patient group taking similar medications to the DILD group but without DILD onset, BP: bacterial pneumonia, NTM: nontuberculous mycobacteriosis, IIPs: idiopathic interstitial pneumonias, CTD: lung disease associated with connective tissue disease, COPD: chronic obstructive pulmonary disease, BA: bronchial asthma.3. Serum IFNγ levels in patients with DILD showing high serum KYN and QUNA levels (n=21), matched pair recovery samples (n=14), and HC (n=18) were measured using a commercially available sandwich ELISA kit.The matched-pair samples between the DILD and DILD recovery groups are indicated by solid lines.The limit of detection (LOD) was 0.469 pg/mL.It has been demonstrated that some patients with DILD showed detectable and elevated serum IFNγ levels, while their levels in all DILD recovery patients and healthy controls were below the detection limit.

Figure S1 .
Figure S1.Comparisons of KYN concentrations between DILD and recovery patients in a validation cohort.The serum concentration of KYN in DILD (n=22) and recovery (n=17) patients in a validation cohort are shown in box-andwhisker plots.The statistical significance between the DILD and recovery groups was tested using the Mann-Whitney U-test.*; p-value < 0.05.

Figure S2 .
Figure S2.Distributions of TRP concentrations in DILD, other lung diseases, and HC.

Figure S3 .
Figure S3.Sensitivity analysis of the sample selection bias.To evaluate sample selection bias, the samples were divided into two subcohorts based on the location of the hospitals (Chiba University and Nippon Medical School; Tokyo metropolitan area [sub-cohort A], Shinshu University and Hiroshima University; and the other area [sub-cohort B], Additional file 1: TableS1).Sub-cohort A included 41 DILD patients and 28 recovery patients, whereas sub-cohort B included 40 DILD patients and 25 recovery patients.A KYN, B QUNA, C TRP concentrations, D KYN/TRP ratio in each sub-cohort are shown in box-and-whisker plots.The statistical significance between the DILD and recovery groups in each sub-cohort was tested using the Mann-Whitney U-test.*; p-value < 0.05, **; p-value < 0.01, ***; p-value < 0.001, ****; pvalue < 0.0001.Samples with missing values were excluded from the statistical analyses.

A
Figure S4.Comparisons of the identified biomarker levels by underlying disease and type of medication for DILD.A The association of the existence of cancers with the serum concentrations of KYN and QUNA, and KYN/TRP ratio in all-DILD patients was analyzed.DILD patients were divided into three groups based on the existence of cancers as underlying diseases.The metabolites concentrations in each group are shown as a box-and-whisker plot.The statistical significances were examined by Mann-Whitney U-test with Bonferroni correction.B Evaluation of the effect of the existence of lifestyle-related diseases on the serum concentrations KYN and QUNA, and KYN/TRP ratio in all-DILD patients were analyzed.DILD patients were divided into subgroups based on the existence of lifestyle-related diseases (cardiovascular diseases, dyslipidemia, hypertension and diabetes).In case one patient have two different lifestyle-related diseases, the data were assigned to both groups.The metabolite concentrations in each group are shown as a box-and-whisker plot.The statistical significances were tested by Mann-Whitney Utest with Bonferroni correction.C The association of types of medications with the serum concentrations of KYN and QUNA, and KYN/TRP ratio in all-DILD patients was analyzed.DILD patients were divided into subgroups based on types of medications.If a patient had two different types of medications, the data were assigned to both groups.The metabolite concentrations in each group are shown as a box-and-whisker plot.The statistical significances were tested by Mann-Whitney U-test with Bonferroni correction.DDAs: DNA damaging agents, ICIs: immune checkpoint inhibitors, TKIs: tyrosine kinase inhibitors.

Figure S5 .A
Figure S5.Correlation of KYN/TRP ratio with severity and mortality of DILD.A Correlation analysis of KYN/TRP ratio with SpO 2 /FiO 2 ratio in acute phase DILD patients (n=44).B Comparison of serum KYN/TRP ratio between DILD patients who survived and those who died due to DILD.The DILD patients who died from causes unrelated to DILD were excluded from the analysis.The statistical significances were tested by Mann-Whitney U-test.ns; not significant.

Figure S7 .
Figure S7.Diagnostic potentials of KYN, QUNA, KYN/TRP ratio, and conventional DILD biomarkers in DAD/DAD-mixed patients.ROC curve analyses of serum levels of KYN and QUNA, KYN/TRP ratio, and levels of conventional ILD biomarkers (SP-D and KL-6) were performed between the groups using the quantitative data in the combined cohort.The ROC curves of DAD/DADmixed patients compared with DILD-tolerant A, DILD recovery B, IIPs C, or CTD D are shown.The values of AUROC are described in the parentheses of the labels for each tested biomarker.The AUROC values for other comparisons are summarized in Table3.

Figure S8 .Figure S9 .
Figure S8.Effect of various inflammatory and anti-inflammatory stimuli on IDO1 mRNA expression in differentiated macrophage cell lines and a lung endothelial cell line.Fold changes of IDO1 mRNA expression levels upon various inflammatory and anti-inflammatory stimuli (10 ng/mL for all cytokines) were examined in differentiated macrophage cell lines (A dTHP1 and B dU937) and lung ECs (C HULEC-5a).The error bar represents the mean ± standard deviation of three independent experiments.Statistical significance of IDO1 mRNA levels between control cells and cytokine-treated cells was tested using Student's t-test with Bonferroni correction (ns, not significant; **, adjusted p-value < 0.01; ***, adjusted p-value < 0.001).